Quiescent Fibroblasts Are More Active in Mounting Robust Inflammatory Responses Than Proliferative Fibroblasts

Abstract

Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E2. As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.

A novel cell-cycle-indicator, mVenus-p27K−, identifies quiescent cells and visualizes G0–G1 transition

“…For example, Idh1, which is responsible for NADPH production, is a key metabolic enzyme in quiescent human fibroblast3, and Nrf2, which works as an important factor in protection from oxidative stress, has recently been reported to play a pivotal role in maintaining hematopoietic stem cells44. As reported4, elevated immune response is one of the characteristics of quiescence. Thus, our mVenus-p27K − marker gives a molecular basis for substantial differences between the cells in the G0 and G1 phases, indicating the feasibility of our system.…”

Quiescent and Proliferative Fibroblasts Exhibit Differential p300 HAT Activation through Control of 5-Methoxytryptophan Production

“…Human fibroblasts are commonly used as a cell model to investigate genetic and biochemical changes accompanying all cycle progression [1]–[3]. We prepared proliferative and quiescent Fb as previously described [3], [4].…”

“…These cellular and metabolic features of quiescent fibroblasts imply that quiescent cells may have specific cellular functions. Our recent report reveals that quiescent fibroblasts are more active in mounting robust inflammatory responses than proliferative fibroblasts [3]. Quiescent fibroblasts express abundant COX-2 proteins in response to phorbol 12-myristate 13-acetate (PMA), interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) and lipopolysaccharide (LPS) [4].…”

Aortic adventitial fibroblast sensitivity to mitogen activated protein kinase inhibitors depends on substrate stiffness

“…Activated AFs can transition toward a migratory myofibroblast phenotype; however, quiescent AFs can also produce signals to recruit and activate leukocytes [4, 5]. In our study, AFs in 10% hydrogels were less proliferative and fewer were positive for αSMA than cells in softer gels.…”

“…Recently, the vascular adventitia has been identified as a critical mediator of arterial disease progression, with adventitial fibroblasts (AFs) implicated in the maladaptive cascades occurring in primary arterial disease and in vessel remodeling and restenosis after vascular intervention [1]. AFs can be activated to differentiate into migratory myofibroblasts [2], to secrete a number of cytokines, chemokines, growth factors, and proteases that affect vascular pathobiology and the behaviors of other resident adventitial cells [3], or to become quiescent and produce inflammatory mediators associated with the accumulation of activated vascular macrophage in diseased vessels [4, 5]. The actions of activated AFs further relate to the progressive extracellular matrix (ECM) remodeling [6, 7] and tissue stiffening [8, 9] that are hallmarks of vascular disease.…”